The Shigella outer membrane autotransporter protein IcsA is localized to the bacterial old pole, where it mediates assembly of an actin tail that propels the bacterium through the intestinal epithelium during infection. IcsA is critical to Shigella virulence. Shigella is estimated to cause 1.1 million deaths annually worldwide and is a Category B Priority Pathogen. In the last decade, work by several investigators has shown that many bacterial proteins, including many involved in virulence, are localized to the pole. During the current funding period, we have made substantial progress in the understanding of polar localization of IcsA. We have shown that polar positional information recognized by IcsA is conserved and is present at or near division sites independent of the factors that are known to control division site selection. These findings are significant because they indicate that insights into positional information recognized by IcsA may provide insights into midcell positional information and mechanisms of localization of other polar proteins. The Aims of this R01 competitive renewal are: 1. Identification and characterization of suppressors of de-localized IcsA derivatives;2. Identification and characterization of the role in IcsA localization of proteins that localize to sites of future poles independent of the cell division machinery;3. Characterization of the role of YidC in proper positioning of polar information;and, 4. Analysis of whether polar secretion is common among large autotransporters. Within these aims, we will apply our results to the analysis of the role of IcsA polarity in Shigella pathogenesis. The approaches we propose are designed to identify and characterize positional information that is recognized by IcsA at the pole. We believe that characterization of the mechanism by which IcsA is localized to the pole will provide insight into the fundamental mechanisms of polar localization of other polar proteins and of positioning of proteins in prokaryotes in general.